Collaborative Research: RUI: Structure-Function Relationships and Efficiency of Bacterial Flagellar Motors Using Computational Fluid Dynamics and Directed Evolution Experiments

细菌是地球上最古老的生物之一。通过生物进化，许多细菌获得了由螺旋附属物组成的生物力学鞭毛，分子马达旋转该附属物使它们在流体环境中移动。鞭毛的遗传组织、调控和结构在细菌物种中广泛保守，鞭毛在细菌的生命周期中发挥着重要作用，包括宿主与微生物的相互作用。因此，了解细菌的运动性在医学、生物学和微型机器人游泳者的发展中具有广泛的意义。本研究旨在了解细菌马达的能量效率如何影响细菌运动系统的进化发展。该项目将创建精确校准的计算工具，以研究具有不同运动特性的医学重要生物体铜绿假单胞菌的变种。将测量每种变种的能效，并将其与发动机部件的结构变化相关联。这些运动变种的计算工具和库将公开提供，以便其他研究人员可以将它们用于相关研究。这项工作所需的实验和计算都将与本科生一起进行，目标是他们中的许多人，包括来自代表性不足的背景的学生，将从事跨学科的科学研究。最近的工作揭示了负责在细菌鞭毛马达中产生扭矩的定子单元的分子结构。本项目旨在表征细菌定子的特定结构属性与其能源效率之间的关系。具有次优定子的铜绿假单胞菌菌株将被创造出来，并用于定向进化实验，以选择具有更高能动性的变异菌株。定量显微镜将用于测量细菌在不同流体环境中移动时的运动。实验确定的轨迹将被输入到精确校准的计算流体力学模拟中，以确定定子的能效。确定具有不同能量效率的进化定子的序列和结构特征将有助于深入了解纳米电机的机械性能和生物分子结构之间的关系。此外，为了研究附近边界的存在如何影响进化菌株的游泳效率，将在光滑表面和具有可调粘度和静电属性的表面附近进行运动性实验。这些表面将由脂质和蛋白质构成，以模拟铜绿假单胞菌在经历从运动到静止的转变时通常在其自然环境中遇到的环境。校准测量将允许其他研究人员创建额外的精确校准的计算流体力学模型，并开发与该项目中使用的类似的计算探测器。公开提供的进化运动变异库和定子的结构-功能图将加深对运动蛋白和运动性能之间关系的理解。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Bacteria are among the oldest organisms on Earth. Through biological evolution, many bacteria have acquired a biomechanical flagellum consisting of a helical appendage that is rotated by a molecular motor to move them through their fluid environment. Flagellar genetic organization, regulation, and structure are broadly conserved across bacterial species, and flagella play vital roles in the bacterial life cycle, including in host-microbe interactions. Thus, understanding bacterial motility has broad implications in medicine, in biology, and in the development of micro-robotic swimmers. This research aims to understand how the energy efficiency of the bacterial motor has influenced the evolutionary development of the bacterial motility system. The project will create precisely calibrated computational tools to study variants of the medically important organism Pseudomonas aeruginosa with different motor properties. The energy efficiency of each variant will be measured and associated with structural changes in motor components. The computational tools and a library of these motor variants will be made publicly available so that other researchers may use them for related research. The experiments and computations required for this work will all be conducted in tandem with undergraduate students, with the goal that many of them, including students from underrepresented backgrounds, will pursue careers in interdisciplinary scientific research.Recent work has uncovered the molecular structure of the stator unit responsible for generating torque in the bacterial flagellar motor. This project aims to characterize the relationships between specific structural properties of the bacterial stator and its energy efficiency. Strains of P. aeruginosa with suboptimal stators will be created and used in directed evolution experiments to select for variant strains with improved motility. Quantitative microscopy will be used to measure bacterial motion as they move through different fluid environments. Experimentally determined trajectories will be input into precisely calibrated computational fluid dynamics simulations to determine the energy efficiency of the stator. The determination of sequence and structural features of evolved stators with different energy efficiencies will provide insights into the relationship between the mechanical properties and biomolecular structures of nanomotors. Additionally, to study how the presence of a nearby boundary affects the swimming efficiency of the evolved strains, motility experiments will be performed near smooth surfaces and surfaces with tunable viscosity and electrostatic properties. The surfaces will be constructed from lipids and proteins to mimic the environments that P. aeruginosa commonly encounters in its natural environment as it undergoes motile-to-sessile transitions. The calibration measurements will allow other researchers to create additional precisely calibrated computational fluid dynamics models and develop similar computational probes as used in this project. The publicly available library of evolved motor variants and the structure-function map for the stator will deepen the understanding of the relationship between motor proteins and motor performance.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.